Adenoviruses have a double-stranded, linear DNA genome of about 36 kb. Adenovirus particles are nonenveloped, icosahedral structures composed of multiple copies of each of twelve virus-encoded proteins and a single copy of the DNA genome. Adenoviruses for many different eukaryotic species have been identified. All adenoviruses have a similar genome organization and virion structure, but adenoviruses from one species do not productively infect the cells of another species. ("The Adenoviruses" Ginsberg, ed, New York: Plenum Press, 1984).
The most extensively studied adenoviruses are the human adenoviruses. At least 46 distinct human adenovirus serotypes have been described. These are classified into six subgroups (A-F) on the basis of related biological and physical properties. The most extensively studied human serotypes are types 2 and 5, which belong to subgroup C. Adenovirus type 2 (Ad2) and type 5 (Ad5) are very closely related; the proteins of Ad2 and Ad5 typically differ at only a few amino acid residues. Adenovirus 12 belongs to subgroup A. Subgroup A adenoviruses differ sufficiently from subgroup C viruses in that corresponding gene products of Ad2 normally will not substitute for the Ad12 product, and vice versa.
All adenovirus genomes are believed to encode closely related proteinases (also called proteases and here, also appropriately called endoproteinases), which cleave several virion precursor polypeptides during virion maturation (Krausslich et al., Ann. Rev. Biochem. 7:701-754 (1988)). The adenovirus endoproteinase (EP) is assembled into virions, with only a few copies in each virion. The adenovirus 2 (Ad2) endoproteinase has been shown to cleave six of the twelve proteins from which virions are assembled. The mature virion components resulting from these six precursors have been designated IIIa, VI, VII (major core protein), VIII, Mu, and TP (terminal protein) (Anderson et al., Virology 172:506-512 (1989)). (The full-length initial products of translation are designated with a p prefix; the C-terminal products of endoproteolytic cleavage are designated by a -c suffix.) Precursor processing occurs primarily, if not exclusively, in young, fully assembled virions (Ishibashi et al., Virology 57:409-424 (1974); Lewis et al., Cold Spring Harbor Symp. Quant. Biol. 39:581-590 (1974)). It has been suggested that the endoproteinase is itself processed autocatalytically (Chatterjee et al., Proc. Natl. Acad. Sci. USA 84:714-718 (1987)).
The 23 kilodalton (kDa)Ad2 EP is encoded by a gene that is expressed at late times after virus infection. The endoproteinase gene is located within the L3 family of adenovirus genes. Evidence that the L3 23 kDa open reading frame encodes an endoproteinase was first provided by analysis of the temperature-sensitive Ad2 mutant, H2ts1 (Weber, J. Virol. 17:462-471 (1976)). The H2ts1 mutation changes proline codon 137 of the L3 23 kDa reading frame to a leucine codon (Yeh-Kai et al., J. Mol. Biol. 167:217-222 (1983)). At the non-permissive temperature, virions produced in H2ts1-infected cells assemble efficiently, but contain precursors in place of the mature components found in wild type particles. Such immature virions attach to cells but fail to yield productive infections (Hannan et al., Intervirology 19:213-223 (1983); Mirza et al., Intervirology 13:307-311 (1980)).
Only a few copies of the adenovirus proteinase are present in each virion. As a consequence of the difficulty of obtaining sufficient quantities of this protein from its natural source, the proteinase polypeptide had not been isolated, its enzymatic activity is not fully characterized, nor is the role of the proteinase in virion maturation well understood.